PROJECT SUMMARY The goal of this proposed research is to reveal the negative regulator of AMPK and eventually target this pathway to generate an efficient AMPK activator for treatment of metabolic syndromes. AMPK senses metabolic stress and is a central mediator in maintaining metabolic homeostasis and energy balance. Thus, AMPK activation has become an attractive target for treating metabolic syndromes, including diabetes and cancer. While it has been demonstrated that AMPK activity is tightly regulated by reversible protein phosphorylation, and despite many efforts to identify AMPK kinases, it is still unclear how AMPK is dephosphorylated or inactivated upon recovery from metabolic stress. One of the main reasons for the limited progress in identifying an AMPK phosphatase is because of the promiscuous activity of serine-threonine phosphatases and its specificity is governed by associated proteins. We and others previously identified that PP2A family protein phosphatases are involved in AMPK dephosphorylation. However, the PP2A phosphatase family contains hundreds of possible different complexes. To identify a specific complex that directly dephosphorylates AMPK, using protein mass- spectrometry analysis, we found that protein phosphatase 6 (PP6, a PP2A family phosphatase) regulatory subunit SAPS3 is associated with AMPK. Furthermore, our preliminary data demonstrated that SAPS3/AMPK binding is glucose responsive and required for AMPK dephosphorylation. To evaluate the role of SAPS3 in vivo, we have generated a novel mouse model by flanking the gene encoding SAPS3, ppp6r3, with a loxP sequence that allow us to develop tissue specific knock out of SAPS3. Using recently developed SAPS3 liver-specific knockout mice, we found that deletion of SAPS3 increases AMPK phosphorylation in liver and displays phenotypes similar to AMPK activation in regulation of metabolism and tumorigenesis. Therefore, we propose a series of experiments in this application to address our central hypothesis that SAPS3-containing PP6 phosphatase complex dephosphorylates and inhibits AMPK activity, thereby regulating AMPK-mediated functions. Three specific aims are proposed as follows: 1) elucidating molecular mechanisms underlying AMPK inhibition by SAPS3-containing PP6 complex, 2) determining the biological function of SAPS3 in metabolic/energy homeostasis in vivo via regulation of AMPK using SAPS3 liver specific knockout mice, 3) examining the role of SAPS3 in tumorigenesis via regulation of AMPK in two mouse tumor models. These studies will provide a solid mechanistic basis for AMPK signaling regulated by protein phosphatase and in vivo evidence that AMPK-mediated biological functions are tightly controlled by protein phosphatase. Results from the proposed research will also advance new therapeutic directions by targeting the SAPS3/AMPK interaction, which could be an effective approach to activate AMPK for treating metabolic syndromes.